In a double or dual beam spectrophotometer, a beam of light is split into two beams. In some instruments this is accomplished on a time share basis. Through a system of mirrors, the light beam is directed along a first path in which a sample can be inserted and a second path containing no sample. The first beam path is referred to as the sample beam and the second beam path is referred to as the reference beam. If a sample of material being less than opaque is inserted in the sample beam, part of the energy of the sample beam is absorbed by the sample material. The amount of energy absorbed is a function of the absorbance characteristics of the material in the sample beam and the particular wavelength of the sample beam. If sensitive instrumentation is provided to receive both the sample beam and the reference beam, the difference in energy between the sample beam and the reference beam can be measured. The difference between the two is indicative of the amount of energy absorbed by the sample. Since different light transmissive materials have different absorptive qualities to different wavelengths of light, a pattern representing the absorbance spectrum of the sample may be generated by subjecting the sample to a light beam which continuously varies in wavelength across or "scans" a predetermined light spectrum. Each material's absorbance spectrum is characteristic of that material in much the same manner as fingerprints or voice prints are characteristic of individual human beings. An operator trained to interpret such outputs can use the spectrophotometer for both qualitative and quantitative analysis.
In such a dual beam spectrophotometer, the absorbance of the sample is directly related to the weight per unit area through which the sample beam is passed. Therefore, the level of absorbance can be increased by increasing the sample size or reducing the sample beam area. This relationship takes on particular importance when examining very small size samples (microsamples), as when the quantity available for sampling is limited. If such a small sample is spread over a large beam area so as to increase the sample size, the absorbance may be too small to measure or, if measured and amplified to a point where it can be detected, the signal-to-noise ratio may be so low as to make the output unreliable. Accordingly, to increase absorbance levels when examining microsamples, it is common practice to reduce the sample beam area by placing the sample in a small aperture of an opaque support plate.
Also in such spectrophotometers, the amount of energy in the sample and reference beams changes at different frequencies or wavelengths within a scan because of variations in the optical efficiencies of the apparatus. In order to maintain the beam at constant energy levels, an adjustable slit is included in a portion of the optical train of the spectrophotometer where the beams are contained, i.e., in a common beam portion of the optical train. Since the energy per unit area varies with wavelength for a variety of reasons, the slit is normally programmed to open in the lower energy regions relative to the higher ones to enlarge the area of the common beam and thereby maintain the total energy of the component sample and reference beams at constant levels. Where (1) the slit width is programmed as a function of wavelength, and is narrower than a sample aperture placed in the sample beam as in FIGS. 1-A and B, and (2) no sample is placed in the aperture, the ratio of the intensity of the sample beam to reference beam remains virtually constant. However, at wavelengths where to maintain constant energy levels the slit program dictates a slit width wider than the sample aperture as in FIGS. 1-C and D, energy in the reference beam increases relative to that of the area limited sample beam and the ratio changes. In essence, energy is being removed in the sample beam by the aperture plate but not in the reference beam and the instrument records a decrease in transmittance at such wavelengths even though there is no actual sample absorption. Further, as the slit opens wider and wider (typically with increasing wavelength), the percentage of energy removed by the fixed area sample aperture plate becomes greater and greater. Such an "aperture effect" manifests itself as a variable 100% base line output from the spectrophotometer when no sample is present and affects the background absorption spectrum when a sample is placed in the sample beam. The aperture effect is depicted in FIG. 2 where the base line 16 is shown to drop off radically at the end of the scan spectrum where the slit opens wider than the sample aperture. With such an output, it becomes difficult for even a trained operator to make an accurate interpretation of the spectrum.
The problems of a variable base line are well recognized. Attempts have been made to provide compensation as typified by the recent patent to K. P. George (U.S. Pat. No. 3,986,776) entitled Automatic Baseline Compensator for Optical Absorption Spectroscopy. George teaches apparatus including adjustable signal attenuating means, calibration run means, magnetic tape recorder means, integrating means, and readback means to apply a compensating signal to the signal attenuating means in synchronism with the wavelength scan. While, in theory this apparatus should work for its intended purpose, it can be seen that a step increase is effected in both cost and complexity by its addition.
Therefore, it is the object of the present invention to provide a simple and effective means for restoring a more constant base line to facilitate analysis using dual beam spectrophotometers in the examination of microsamples where small apertures are required to be placed in the sample beam.